CN107354190A - The process of antibacterial peptide is prepared using bacillus licheniformis - Google Patents
The process of antibacterial peptide is prepared using bacillus licheniformis Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, and in particular to a kind of process that antibacterial peptide is prepared using bacillus licheniformis.The antibacterial peptide that the present invention is prepared by the production technology such as the activation of inclined-plane bacterium, Shaking culture, seed culture, fermented and cultured, fermentation liquor pretreatment and spray drying, screening and packaging has higher antibacterial activity; and present invention process cost is low, the large-scale production of antibacterial peptide can be realized.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of work that antibacterial peptide is prepared using bacillus licheniformis
Process.
Background technology
Antibacterial peptide is that have broad-spectrum sterilization through inducing the caused micromolecule polypeptide with bioactivity in a kind of organism
Effect.Natural antibacterial peptide, it has been found that have A, B and D etc. a variety of.This kind of active peptides have preferable resistance to acids and bases, heat steady more
The characteristics such as qualitative and broad-spectrum antiseptic.Antibacterial peptide has very strong lethal effect to bacterium, especially to some drug resistance pathogens
With very strong killing action.It is that its sterilization mechanism is its hydrophobicity compared with conventional antibiotic, the characteristics of antibacterial peptide maximum
End spiral is inserted directly into bacterium mycetocyte film and forms duct, releases extracellular by bacterium content and causes bacterial death, has
Act on the good characteristics that rapid, effective to drug-fast bacteria and bacterium is not likely to produce drug resistance.
Bacillus is the aerobic or amphimicrobian G of one kind+(at present it has also been found that there is G-), it can produce under certain condition degeneration-resistant
The chemoheterotrophic bacteria of property endospore.Very extensive in distributed in nature, physiological property is rich and varied, is soil and the micro- life of plant
One of state dominant population.It can produce Multiple Classes of Antibiotics, including lipopeptid class, peptides, phospholipid, more alkenes, amino acids, nucleic acid
Class material, good inhibiting effect is played to a variety of animal and plant and human pathogen bacterium.And bacillus also has very strong egg
White enzyme, lipase, amylase activity.Therefore, bacillus is widely used in medicine, agricultural chemicals, food, feed manufacturing, environment
The industry-by-industries such as pollution control.Bacillus licheniformis (Bacillus subtilis) be widely present in nature it is a kind of non-
Pathogenic bacteria, a variety of antibacterial materials can be produced during growth metabolism, include the lipopeptid class antibiosis of non-ribosomal synthesis
Element, the lantibiotics and antibacterial protein of Ribosome biogenesis.These antibacterial materials are suppressing bacterium, fungi and yeast pollution side
Face has important application.Also there is important application value in food, medicine, chemical industry and controlling plant diseases etc..It is Chinese special
Sharp (CN103772490B) discloses a kind of antibacterial peptide of bacillus licheniformis secretion and its preparation method and application.Described is anti-
Bacterium peptide amino acid sequence is shown in sequence table SEQ ID NO.1, and molecular weight is 1290.4 dalton, isoelectric point 5.23.It is prepared
Method is：The antibacterial peptide that bacillus licheniformis secretes in ammonium sulfate precipitation culture medium, utilizes anion exchange, sieve chromatography
Antibacterial peptide is purified with RP-HPLC method.The antibacterial peptide has significant antibacterial activity, and will not be to the red blood cell of pig
Haemocylolysis is produced, feeding antibacterials are prepared available for exploitation.
But the report using bacillus licheniformis large scale fermentation production antibacterial peptide is less, and antibacterial activity is low, or even nothing
Antibacterial activity.
The content of the invention
To solve the above problems, the invention provides a kind of process that antibacterial peptide is prepared using bacillus licheniformis.
The present invention passes through the activation of inclined-plane bacterium, Shaking culture, seed culture, fermented and cultured, fermentation liquor pretreatment and spray drying, screening
The antibacterial peptide being prepared with production technologies such as packagings has higher antibacterial activity, and present invention process cost is low, can
Realize the large-scale production of antibacterial peptide.
The present invention is achieved through the following technical solutions：
A kind of process that antibacterial peptide is prepared using bacillus licheniformis, is comprised the following steps：
S1. inclined-plane bacterium activates：Antibacterial peptide production strain Bacillus licheniformis is taken to be seeded on slant medium, 25 DEG C~40
10~30h is cultivated under the conditions of DEG C；
S2. Shaking culture：1~10mL sterilized waters are added into the cultured slant mediums of step S1 and prepare bacteria suspension,
Take 1~3mL bacterial suspension inoculations to the YPD culture mediums containing 50mL shaking flask in, 25 DEG C~35 DEG C, 250r/min culture 15~
25h；YPD culture mediums, by percentage to the quality, it is formulated and is：1~8% dusty yeast, 1~8% peptone, 1~8% glucose, pH
It is natural；
S3. seed culture：After first class seed pot medium sterilization, prepared seed liquor in inoculation step S2；Connecing
Kind amount is 0.1%~2%, and fermentation temperature is 20 DEG C~40 DEG C, and speed of agitator 180-220 turns/min, and ventilation is 10~20L/
Min, dissolved oxygen are 10~50%, and zymotic fluid pH value is that 10~30h is cultivated under 6.0 condition of culture, when residual sugar in seeding tank zymotic fluid
Measure for 0.1~2.0% when stream plus 50% glucose；
S4. fermented and cultured：With the first class seed pot nutrient solution prepared by the inoculum concentration inoculation step S3 of 4~8% volume ratios
It it is 20 DEG C~50 DEG C in fermentation temperature, tank pressure is 0.001~1Mpa, stirring in the 50L fermentation tanks of the fermentation medium containing 20L
Rotating speed is 100~400r/min, and ventilation is 10~30L/min, and dissolved oxygen is to ferment under conditions of 10~50%, pH3.0~8.0
80~100h, when residual sugar amount is 0.01~3.0% in ferment tank liquid, stream plus 50% glucose solution；
S5. fermentation liquor pretreatment：Zymotic fluid to 6.0~6.7 and is heated to 40~60 by regulating step S4 zymotic fluids pH value
DEG C, 50~100mg/L chitosans are added, after floccule body generation, by supernatant after 50r/min stirring 5min, 30~50min of standing
Liquid is first by the milipore filter ultrafiltration that molecular cut off is 2kDa, and after feed liquid concentrates 8~10 times, the filtrate for collecting to obtain passes through again
The milipore filter that molecular cut off is 1000Da is concentrated by ultrafiltration, and obtains concentrate；
S6. it is spray-dried：It is spray-dried filtrate is concentrated made from step S5, obtains antibacterial peptide pulvis；
S7. antibacterial peptide pulvis made from step S6 is subjected to screening packaging, produced.
Preferably, the process that antibacterial peptide is prepared using bacillus licheniformis, is comprised the following steps：
S1. inclined-plane bacterium activates：Antibacterial peptide production strain Bacillus licheniformis is taken to be seeded on slant medium, 35 DEG C of conditions
Lower culture 18h；
S2. Shaking culture：5mL sterilized waters are added into the cultured slant mediums of step S1 and prepare bacteria suspension, are taken
2.5mL bacterial suspension inoculations are into the shaking flask of the YPD culture mediums containing 50mL, 35 DEG C, 250r/min cultures 20h；YPD culture mediums,
By percentage to the quality, it is formulated and is：5% dusty yeast, 5% peptone, 4% glucose, pH are natural；
S3. seed culture：After first class seed pot medium sterilization, prepared seed liquor in inoculation step S2；Connecing
Kind amount is 0.8%, and fermentation temperature is 35 DEG C, 200 turns/min of speed of agitator, ventilation 15L/min, dissolved oxygen 20%, is fermented
Liquid pH value is to cultivate 20h under 6.0 condition of culture, stream plus 50% glucose when residual sugar amount is 1.2% in seeding tank zymotic fluid；
S4. fermented and cultured：With the first class seed pot nutrient solution prepared by the inoculum concentration inoculation step S3 of 5% volume ratio in containing
It is 36 DEG C in fermentation temperature in the 50L fermentation tanks of 20L fermentation mediums, tank pressure is 0.8Mpa, speed of agitator 360r/min,
Ventilation is 25L/min, dissolved oxygen 30%, and ferment 96h under conditions of pH5.5, when residual sugar amount is 1.5% in ferment tank liquid
When, stream adds 50% glucose solution；
S5. fermentation liquor pretreatment：Zymotic fluid to 6.5 and is heated to 55 DEG C by regulating step S4 zymotic fluids pH value, is added
85mg/L chitosans, after floccule body generation, 50r/min stirring 5min, supernatant is divided by retention first after standing 45min
Son amount is 2kDa milipore filter ultrafiltration, and after feed liquid concentrates 9 times, the filtrate for collecting to obtain is again 1000Da's through molecular cut off
Milipore filter is concentrated by ultrafiltration, and obtains concentrate；
S6. it is spray-dried：It is spray-dried filtrate is concentrated made from step S5, obtains antibacterial peptide pulvis；
S7. antibacterial peptide pulvis made from step S6 is subjected to screening packaging, produced.
Preferably, slant medium described in step S1, by percentage to the quality, it is formulated and is：Dusty yeast 1~8%, peptone
1~8%, glucose 1~8%, agar powder 1~8%, pH natures.
Further, slant medium described in step S1, by percentage to the quality, it is formulated and is：Dusty yeast 4.5%, albumen
Peptone 5%, glucose 6%, agar powder 2%, pH are natural.
Preferably, the zeocin in the YPD culture mediums also containing 100 μ g/mL.
Preferably, the step S4 fermentative medium formulas are 1~5g/dL of glucose, 0.5~4g/dL of peptone, yeast
Powder 0.1~2g/dL, NH4H2PO41~10g/dL, K2SO41~10g/dL, MgSO4 7H2O 0.5~5g/dL, KH2PO4 0.1
~3g/dL, CaSO40.01~2g/dL of 0.001~0.5g/dL, KOH 0.5~3g/dL, PTM1.
Further, the step S4 fermentative medium formulas are glucose 3g/dL, peptone 2.5g/dL, dusty yeast
1.5g/dL, NH4H2PO44g/dL, K2SO46g/dL, MgSO4 7H2O 3.5g/dL, KH2PO40.8g/dL, CaSO4
0.15g/dL, KOH 2.5g/dL, PTM1 0.25g/dL.
Preferably, also contain containing 1.2% PTM1 in step S4 streams plus 50% glucose solution.
Compared with prior art, major technique advantage of the invention is：
(1) present invention is made up of Optimal Medium, has been effectively facilitated bacillus licheniformis secretion antibacterial peptide, has been realized anti-
The scale of bacterium peptide, industrialization production；
(2) present invention process produces obtained antibacterial peptide good antimicrobial effect, and property is stable, is established for the popularization and application of antibacterial peptide
Basis is determined.
Embodiment
The present invention is further described in detail below in conjunction with embodiment.It is pointed out that following explanation is only pair
Claimed is technical scheme for example, not to any restrictions of these technical schemes.The protection of the present invention
Scope is defined by the content that appended claims are recorded.
Zeocin is purchased from Shanghai past bio tech ltd；PTM1 is purchased from the limited public affairs of Nanjing Sen Beijia biotechnologies
Department.
A kind of process that antibacterial peptide is prepared using bacillus licheniformis of embodiment 1
The process that antibacterial peptide is prepared using bacillus licheniformis, is comprised the following steps：
S1. inclined-plane bacterium activates：Antibacterial peptide production strain Bacillus licheniformis is taken to be seeded on slant medium, 35 DEG C of conditions
Lower culture 18h；The slant medium, by percentage to the quality, it is formulated and is：Dusty yeast 4.5%, peptone 5%, glucose
6%, agar powder 2%, pH natures；
S2. Shaking culture：5mL sterilized waters are added to the cultured slant mediums of step S1 and prepare bacteria suspension, take 2.5mL
Bacterial suspension inoculation is into the shaking flask of the YPD culture mediums containing 50mL, 35 DEG C, 250r/min cultures 20h；YPD culture mediums, with quality
Percentages, are formulated and are：5% dusty yeast, 5% peptone, 4% glucose, pH are natural；Also contain 100 in the YPD culture mediums
μ g/mL zeocin；
S3. seed culture：After first class seed pot medium sterilization, prepared seed liquor in inoculation step S2；Connecing
Kind amount is 0.8%, and fermentation temperature is 35 DEG C, 200 turns/min of speed of agitator, ventilation 15L/min, dissolved oxygen 20%, is fermented
Liquid pH value is to cultivate 20h under 6.0 condition of culture, stream plus 50% glucose when residual sugar amount is 1.2% in seeding tank zymotic fluid；
S4. fermented and cultured：With the first class seed pot nutrient solution prepared by the inoculum concentration inoculation step S3 of 5% volume ratio in containing
It is 36 DEG C in fermentation temperature in the 50L fermentation tanks of 20L fermentation mediums, tank pressure is 0.8Mpa, speed of agitator 360r/min,
Ventilation is 25L/min, dissolved oxygen 30%, and ferment 96h under conditions of pH5.5, when residual sugar amount is 1.5% in ferment tank liquid
When, stream adds 50% and the glucose solution containing 1.2%PTM1；Fermentative medium formula is glucose 3g/dL, peptone
2.5g/dL, dusty yeast 1.5g/dL, NH4H2PO44g/dL, K2SO46g/dL, MgSO4 7H2O 3.5g/dL, KH2PO4 0.8g/
DL, CaSO40.15g/dL, KOH 2.5g/dL, PTM1 0.25g/dL.
S5. fermentation liquor pretreatment：Zymotic fluid to 6.5 and is heated to 55 DEG C by regulating step S4 zymotic fluids pH value, is added
85mg/L chitosans, after floccule body generation, 50r/min stirring 5min, supernatant is divided by retention first after standing 45min
Son amount is 2kDa milipore filter ultrafiltration, and after feed liquid concentrates 9 times, the filtrate for collecting to obtain is again 1000Da's through molecular cut off
Milipore filter is concentrated by ultrafiltration, and obtains concentrate；
S6. it is spray-dried：Concentrate made from step S5 is spray-dried, obtains antibacterial peptide pulvis；
S7. antibacterial peptide pulvis made from step S6 is subjected to screening packaging, produced.
A kind of process that antibacterial peptide is prepared using bacillus licheniformis of embodiment 2
The process that antibacterial peptide is prepared using bacillus licheniformis, is comprised the following steps：
S1. inclined-plane bacterium activates：Antibacterial peptide production strain Bacillus licheniformis is taken to be seeded on slant medium, 25 DEG C of conditions
Lower culture 10h；The slant medium, by percentage to the quality, it is formulated and is：Dusty yeast 1%, peptone 8%, glucose 8%,
Agar powder 1%, pH are natural；
S2. Shaking culture：1mL sterilized waters are added to the cultured slant mediums of step S1 and prepare bacteria suspension, take 1mL bacterium
Suspension is seeded in the shaking flask of the YPD culture mediums containing 50mL, 25 DEG C, 250r/min cultures 15h；YPD culture mediums, with quality hundred
Point than meter, it is formulated and is：1% dusty yeast, 8% peptone, 8% glucose, pH are natural；Also contain 100 μ in the YPD culture mediums
G/mL zeocin；
S3. seed culture：After first class seed pot medium sterilization, prepared seed liquor in inoculation step S2；Connecing
Kind amount is 0.1%, and fermentation temperature is 20 DEG C, 180 turns/min of speed of agitator, ventilation 10L/min, dissolved oxygen 10%, is fermented
Liquid pH value is to cultivate 10h under 6.0 condition of culture, stream plus 50% glucose when residual sugar amount is 0.1% in seeding tank zymotic fluid；
S4. fermented and cultured：With the first class seed pot nutrient solution prepared by the inoculum concentration inoculation step S3 of 4% volume ratio in containing
It it is 20 DEG C in fermentation temperature, tank pressure is 0.001Mpa, speed of agitator 100r/ in the 50L fermentation tanks of 20L fermentation mediums
Min, ventilation 10L/min, dissolved oxygen 10%, ferment under conditions of pH3.0 80h, when residual sugar amount is in ferment tank liquid
When 0.01%, stream plus 50% and the glucose solution containing 1.2%PTM1；Fermentative medium formula is glucose 1g/dL, albumen
Peptone 4g/dL, dusty yeast 0.1g/dL, NH4H2PO410g/dL, K2SO41~g/dL, MgSO4 7H2O 0.5g/dL, KH2PO4
3g/dL, CaSO40.001g/dL, KOH 3g/dL, PTM1 0.01g/dL；
S5. fermentation liquor pretreatment：Zymotic fluid to 6.0 and is heated to 40 DEG C by regulating step S4 zymotic fluids pH value, is added
50mg/L chitosans, after floccule body generation, 50r/min stirring 5min, supernatant is divided by retention first after standing 30min
Son amount is 2kDa milipore filter ultrafiltration, and after feed liquid concentrates 8 times, the filtrate for collecting to obtain is again 1000Da's through molecular cut off
Milipore filter is concentrated by ultrafiltration, and obtains concentrate；
S6. it is spray-dried：It is spray-dried filtrate is concentrated made from step S5, obtains antibacterial peptide pulvis；
S7. antibacterial peptide pulvis made from step S6 is subjected to screening packaging, produced.
A kind of process that antibacterial peptide is prepared using bacillus licheniformis of embodiment 3
A kind of process that antibacterial peptide is prepared using bacillus licheniformis, is comprised the following steps：
S1. inclined-plane bacterium activates：Antibacterial peptide production strain Bacillus licheniformis is taken to be seeded on slant medium, 40 DEG C of conditions
Lower culture 30h；The slant medium, by percentage to the quality, it is formulated and is：Dusty yeast 8%, peptone 1%, glucose 1%,
Agar powder 1%, pH are natural；
S2. Shaking culture：10mL sterilized waters are added into the cultured slant mediums of step S1 and prepare bacteria suspension, are taken
3mL bacterial suspension inoculations are into the shaking flask of the YPD culture mediums containing 50mL, 35 DEG C, 250r/min cultures 25h；YPD culture mediums, with
Mass percent meter, is formulated and is：8% dusty yeast, 1% peptone, 8% glucose, pH are natural；Also contain in the YPD culture mediums
There is 100 μ g/mL zeocin；
S3. seed culture：After first class seed pot medium sterilization, prepared seed liquor in inoculation step S2；Connecing
Kind of amount is 2%, and fermentation temperature is 40 DEG C, speed of agitator 220 turns/min, ventilation 20L/min, dissolved oxygen 50%, zymotic fluid
PH value is to cultivate 30h under 6.0 condition of culture, stream plus 50% glucose when residual sugar amount is 2.0% in seeding tank zymotic fluid；
S4. fermented and cultured：With the first class seed pot nutrient solution prepared by the inoculum concentration inoculation step S3 of 8% volume ratio in containing
It it is 50 DEG C in fermentation temperature, tank pressure is 1Mpa, speed of agitator 400r/min, is led in the 50L fermentation tanks of 20L fermentation mediums
Air quantity is 30L/min, dissolved oxygen 50%, and ferment 100h under conditions of pH8.0, when residual sugar amount is 3.0% in ferment tank liquid
When, stream adds 50% and the glucose solution containing 1.2%PTM1；Fermentative medium formula is glucose 5g/dL, peptone
0.5g/dL, dusty yeast 2g/dL, NH4H2PO41g/dL, K2SO410g/dL, MgSO4 7H2O 5g/dL, KH2PO40.1g/dL,
CaSO40.5g/dL, KOH 0.5g/dL, PTM1 2g/dL；
S5. fermentation liquor pretreatment：Zymotic fluid to 6.7 and is heated to 60 DEG C by regulating step S4 zymotic fluids pH value, is added
100mg/L chitosans, after floccule body generation, 50r/min stirring 5min, supernatant is passed through to retention first after standing 50min
Molecular weight is 2kDa milipore filter ultrafiltration, and after feed liquid concentrates 8~10 times, the filtrate for collecting to obtain is again through molecular cut off
1000Da milipore filter is concentrated by ultrafiltration, and obtains concentrate；
S6. it is spray-dried：It is spray-dried filtrate is concentrated made from step S5, obtains antibacterial peptide pulvis；
S7. antibacterial peptide pulvis made from step S6 is subjected to screening packaging, produced.
A kind of process that antibacterial peptide is prepared using bacillus licheniformis of comparative example 1
Difference with embodiment 1 is that step S4 fermentation mediums are LB culture mediums：Tryptone 10g/L, yeast extraction
Thing 5g/L, sodium chloride 10g/L；Other steps are similar to Example 1.
A kind of process that antibacterial peptide is prepared using bacillus licheniformis of comparative example 2
Difference with embodiment 1 is that dissolved oxygen is 60% in the step S4, and other steps are similar to Example 1.
The antibacterial peptide Determination of Antibacterial Activity of test example 1
(1) subjects：It is utilized respectively the antibacterial peptide that embodiment 1~3 and the process of comparative example 1~2 are prepared；
(2) test method：
The measure of antibacterial activity uses minimal inhibitory concentration (MIC) method, and it is false to Escherichia coli, verdigris to detect antibacterial peptide respectively
Monad, the antibacterial activity of staphylococcus aureus, method are as follows：First add what 90 μ L were prepared into sterile 96 hole round bottom plate
Bacteria suspension, then add the antibacterial peptide dilution of 10 μ L respective concentrations one by one, the final concentration of peptide to be measured is respectively 256,128,64,
32、16、8、4、2、1、0.5、0.25μg/mL；Positive control wells are set in addition to be not added with antibacterial material, only add 100 μ L bacteria suspensions, it is cloudy
Property control wells add 100 μ LMH broth bouillons；It is small that 96 orifice plates are placed in moisturizing quiescent culture 18-24 in 37 DEG C of biochemical cultivation cases
When；Every kind of antibacterial material is done to be repeated three times；Whether each bottom hole portion of observation has bacterial precipitation generation after having cultivated, and is visible by naked eyes thin
The Cmin of bacterium precipitation can determine that the MIC for antibacterial material,
(3) result of the test：Test as shown in table 1 below；
The minimal inhibitory concentration of the micro-broth dilution method difference antibacterial peptide of table 1
As shown in Table 1, the fungistatic effect of antibacterial peptide is superior to pair made from the processing of the process of the embodiment of the present invention 1~3
Ratio 1 and comparative example 2.
The Detection of Stability of the antibacterial peptide of test example 2
(1) heat stability test of antibacterial peptide
(1) subjects：It is utilized respectively the antibacterial peptide that embodiment 1~3 and the process of comparative example 1~2 are prepared；
(2) test method：1g antibacterial peptide products are weighed respectively, are dissolved in respectively in 50mL water, different temperatures gradient water-bath
After middle insulation 20 minutes, its antibacterial activity is determined.
(3) result of the test：As shown in table 2.
Antibacterial peptide vigor under the different temperatures of table 2
As shown in Table 2, the antibacterial peptide that the embodiment of the present invention 1~3 is prepared has preferable heat endurance, in temperature
After 100 DEG C of water-bath 20min, Antibacterial Activity is more than 90%.
(2) the enzyme stability detection of antibacterial peptide
(1) subjects：It is utilized respectively the antibacterial peptide that embodiment 1~3 and the process of comparative example 1~2 are prepared；
(2) test method：Weigh 1g antibacterial peptide products respectively, pH=3.0, under the conditions of 37 DEG C, simulate in animal stomach
The concentration level of enzyme, pepsin, trypsase, protease hydrolyzed processing antibacterial peptide 2h is respectively adopted, then determines antibacterial peptide
Inhibitory potency.In continuous mode, it is dissolved in respectively in 50mL water using clear water processing as control, in different temperatures gradient water-bath
After insulation 20 minutes, its antibacterial activity is determined.
(3) result of the test：As shown in table 3.
Antibacterial peptide activity after 3 different ferment treatments of table
As shown in Table 3, the antibacterial peptide activity that the embodiment of the present invention 1~3 is prepared is after digesting ferment treatment, under enzyme activity
Simultaneously unobvious are dropped, thus illustrate that antibacterial peptide of the present invention has preferable enzyme stability.
Claims (8)
1. a kind of process that antibacterial peptide is prepared using bacillus licheniformis, it is characterised in that comprise the following steps：
S1. inclined-plane bacterium activates：Antibacterial peptide production strain Bacillus licheniformis is taken to be seeded on slant medium, 25 DEG C~40 DEG C bars
10~30h is cultivated under part；
S2. Shaking culture：1~10mL sterilized waters are added into the cultured slant mediums of step S1 and prepare bacteria suspension, take 1~
3mL bacterial suspension inoculations are into the shaking flask of the YPD culture mediums containing 50mL, 25 DEG C~35 DEG C, 15~25h of 250r/min cultures；YPD
Culture medium, by percentage to the quality, it is formulated and is：1~8% dusty yeast, 1~8% peptone, 1~8% glucose, pH are natural；
S3. seed culture：After first class seed pot medium sterilization, prepared seed liquor in inoculation step S2；In inoculum concentration
For 0.1%~2%, fermentation temperature is 20 DEG C~40 DEG C, and speed of agitator 180-220 turns/min, and ventilation is 10~20L/min,
Dissolved oxygen is 10~50%, and zymotic fluid pH value is that 10~30h is cultivated under 6.0 condition of culture, when residual sugar amount is in seeding tank zymotic fluid
Stream plus 50% glucose when 0.1~2.0%；
S4. fermented and cultured：With the first class seed pot nutrient solution prepared by the inoculum concentration inoculation step S3 of 4~8% volume ratios in containing
It it is 20 DEG C~50 DEG C in fermentation temperature, tank pressure is 0.001~1Mpa, speed of agitator in the 50L fermentation tanks of 20L fermentation mediums
For 100~400r/min, ventilation is 10~30L/min, dissolved oxygen be fermentation 80 under conditions of 10~50%, pH3.0~8.0~
100h, when residual sugar amount is 0.01~3.0% in ferment tank liquid, stream plus 50% glucose solution；
S5. fermentation liquor pretreatment：Zymotic fluid to 6.0~6.7 and is heated to 40~60 DEG C by regulating step S4 zymotic fluids pH value, is added
Enter 50~100mg/L chitosans, it is after 50r/min stirring 5min, 30~50min of standing that supernatant is first after floccule body generation
The milipore filter ultrafiltration that molecular cut off is 2kDa is first passed through, after feed liquid concentrates 8~10 times, the filtrate for collecting to obtain is again through retention
The milipore filter that molecular weight is 1000Da is concentrated by ultrafiltration, and obtains concentrate；
S6. it is spray-dried：It is spray-dried filtrate is concentrated made from step S5, obtains antibacterial peptide pulvis；
S7. antibacterial peptide pulvis made from step S6 is subjected to screening packaging, produced.
2. the process of antibacterial peptide is prepared using bacillus licheniformis according to claim 1, it is characterised in that including such as
Lower step：
S1. inclined-plane bacterium activates：Take antibacterial peptide production strain Bacillus licheniformis to be seeded on slant medium, trained under the conditions of 35 DEG C
Support 18h；
S2. Shaking culture：5mL sterilized waters are added into the cultured slant mediums of step S1 and prepare bacteria suspension, take 2.5mL bacterium
Suspension is seeded in the shaking flask of the YPD culture mediums containing 50mL, 35 DEG C, 250r/min cultures 20h；YPD culture mediums, with quality hundred
Point than meter, it is formulated and is：5% dusty yeast, 5% peptone, 4% glucose, pH are natural；
S3. seed culture：After first class seed pot medium sterilization, prepared seed liquor in inoculation step S2；In inoculum concentration
For 0.8%, fermentation temperature is 35 DEG C, speed of agitator 200 turns/min, ventilation 15L/min, dissolved oxygen 20%, zymotic fluid pH
It is worth to cultivate 20h under 6.0 condition of culture, stream plus 50% glucose when residual sugar amount is 1.2% in seeding tank zymotic fluid；
S4. fermented and cultured：With the first class seed pot nutrient solution prepared by the inoculum concentration inoculation step S3 of 5% volume ratio in containing 20L
It it is 36 DEG C in fermentation temperature, tank pressure is 0.8Mpa, speed of agitator 360r/min, is divulged information in the 50L fermentation tanks of fermentation medium
Measuring as 25L/min, dissolved oxygen 30%, ferment 96h under conditions of pH5.5, when residual sugar amount is 1.5% in ferment tank liquid,
Stream plus 50% glucose solution；
S5. fermentation liquor pretreatment：Zymotic fluid to 6.5 and is heated to 55 DEG C by regulating step S4 zymotic fluids pH value, adds 85mg/L
Chitosan, after floccule body generation, it is by molecular cut off first by supernatant after 50r/min stirring 5min, standing 45min
2kDa milipore filter ultrafiltration, after feed liquid concentrates 9 times, obtained filtrate is collected again through milipore filter that molecular cut off is 1000Da
It is concentrated by ultrafiltration, obtains concentrate；
S6. it is spray-dried：It is spray-dried filtrate is concentrated made from step S5, obtains antibacterial peptide pulvis；
S7. antibacterial peptide pulvis made from step S6 is subjected to screening packaging, produced.
3. the process according to claim 1 or claim 2 that antibacterial peptide is prepared using bacillus licheniformis, it is characterised in that step
Slant medium described in rapid S1, by percentage to the quality, it is formulated and is：Dusty yeast 1~8%, peptone 1~8%, glucose 1~
8%, agar powder 1~8%, pH natures.
4. the process of antibacterial peptide is prepared using bacillus licheniformis according to claim 3, it is characterised in that step S1
The slant medium, by percentage to the quality, it is formulated and is：Dusty yeast 4.5%, peptone 5%, glucose 6%, agar powder
2%, pH are natural.
5. the process according to claim 1 or claim 2 that antibacterial peptide is prepared using bacillus licheniformis, it is characterised in that institute
State the zeocin also containing 100 μ g/mL in YPD culture mediums.
6. the process according to claim 1 or claim 2 that antibacterial peptide is prepared using bacillus licheniformis, it is characterised in that institute
It is 1~5g/dL of glucose, 0.5~4g/dL of peptone, 0.1~2g/dL of dusty yeast to state step S4 fermentative medium formulas,
NH4H2PO41~10g/dL, K2SO41~10g/dL, MgSO4 7H2O 0.5~5g/dL, KH2PO40.1~3g/dL, CaSO4
0.01~2g/dL of 0.001~0.5g/dL, KOH 0.5~3g/dL, PTM1.
7. the process of antibacterial peptide is prepared using bacillus licheniformis according to claim 6, it is characterised in that the step
Rapid S4 fermentative medium formulas are glucose 3g/dL, peptone 2.5g/dL, dusty yeast 1.5g/dL, NH4H2PO44g/dL,
K2SO46g/dL, MgSO4 7H2O 3.5g/dL, KH2PO40.8g/dL, CaSO40.15g/dL, KOH 2.5g/dL, PTM1
0.25g/dL。
8. the process according to claim 1 or claim 2 that antibacterial peptide is prepared using bacillus licheniformis, it is characterised in that step
Also contain containing 1.2% PTM1 in rapid S4 streams plus 50% glucose solution.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107412732A (en) * | 2017-07-24 | 2017-12-01 | 广东容大生物股份有限公司 | A kind of application of antibacterial peptide in scar repair medicine is prepared |
| CN108998486A (en) * | 2018-08-02 | 2018-12-14 | 江南大学 | A kind of albuminiferous abductive approach of xylose inducible bacillus licheniformis |
| CN113481267A (en) * | 2021-07-05 | 2021-10-08 | 武汉华扬天乐动物保健品有限公司 | Antibacterial peptide fermentation process |
| CN114478721A (en) * | 2022-02-09 | 2022-05-13 | 安杰利(重庆)生物科技有限公司 | Method for large-scale production of lasso peptide 21 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899375A (en) * | 2012-09-20 | 2013-01-30 | 天津科技大学 | Method for increasing titer of bacitracin in fermentation liquid by using oxygen carrier |
| CN103772490A (en) * | 2014-01-18 | 2014-05-07 | 浙江大学 | Antibacterial peptide secreted by bacillus licheniformis, preparation method and application thereof |
| CN104928202A (en) * | 2014-03-21 | 2015-09-23 | 中国科学院沈阳应用生态研究所 | Fermentation culture method of bacillus |
| CN105483064A (en) * | 2016-02-23 | 2016-04-13 | 华北制药集团新药研究开发有限责任公司 | Method for fermentation production of bacitracin |
| CN106282276A (en) * | 2015-05-18 | 2017-01-04 | 北京伟嘉人生物技术有限公司 | A kind of preparation method and application of bacillus cereus antibacterial peptide |
-
2017
- 2017-07-24 CN CN201710608545.4A patent/CN107354190A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899375A (en) * | 2012-09-20 | 2013-01-30 | 天津科技大学 | Method for increasing titer of bacitracin in fermentation liquid by using oxygen carrier |
| CN103772490A (en) * | 2014-01-18 | 2014-05-07 | 浙江大学 | Antibacterial peptide secreted by bacillus licheniformis, preparation method and application thereof |
| CN104928202A (en) * | 2014-03-21 | 2015-09-23 | 中国科学院沈阳应用生态研究所 | Fermentation culture method of bacillus |
| CN106282276A (en) * | 2015-05-18 | 2017-01-04 | 北京伟嘉人生物技术有限公司 | A kind of preparation method and application of bacillus cereus antibacterial peptide |
| CN105483064A (en) * | 2016-02-23 | 2016-04-13 | 华北制药集团新药研究开发有限责任公司 | Method for fermentation production of bacitracin |
Non-Patent Citations (3)
| Title |
|---|
| 喻钢等: "枯草芽孢杆菌抗菌肽的分离纯化及质谱检测", 《药物分析杂志》 * |
| 崔京春等: "地衣芽孢杆菌发酵过程的优化", 《饲料博览》 * |
| 邓坤等: "溶氧对地衣芽孢杆菌DW2合成杆菌肽的影响", 《中国抗生素杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107412732A (en) * | 2017-07-24 | 2017-12-01 | 广东容大生物股份有限公司 | A kind of application of antibacterial peptide in scar repair medicine is prepared |
| CN108998486A (en) * | 2018-08-02 | 2018-12-14 | 江南大学 | A kind of albuminiferous abductive approach of xylose inducible bacillus licheniformis |
| CN113481267A (en) * | 2021-07-05 | 2021-10-08 | 武汉华扬天乐动物保健品有限公司 | Antibacterial peptide fermentation process |
| CN114478721A (en) * | 2022-02-09 | 2022-05-13 | 安杰利(重庆)生物科技有限公司 | Method for large-scale production of lasso peptide 21 |
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